Caspases are the cysteine proteases that control apoptotic cell death. If caspases can be activated, cancer cells die; conversely inhibiting caspases could prevent cell death in diseases like heart attack and stroke. Thus there has been significant interest in caspases as drug targets. This interest was heightened when caspase-6 was discovered to play a central role in neurodegenerative diseases. Unfortunately, to date, no caspase-directed therapies are on the market, primarily because work has focused on targeting the active site, which is the most overlapping and conserved region of the family. It is becoming increasingly clear that each caspase is regulated in a unique and complex manner so the most promising avenue for achieving caspase-specific inhibition may be by harnessing allosteric sites. In order to target a specific caspase or group of caspases allosterically, it is essential to understand the differences between individual caspases and the similarities within subgroups in the caspase family. Thus, the goal of this project is understand how phosphorylation and zinc contribute to regulation of caspase activity. Understanding the roles of phosphorylation or zinc binding alone provides critical information about natural regulatory processes for each member of the apoptotic caspases. Together these sites highlight key sensitive regions that allow strategic control of caspase function. Caspases are extensively phosphorylated. Most phosphorylation events lead to inactivation of caspase function. Our first approach uses methods we have developed for structural analysis of phosphomimetic and phosphorylated versions of caspases. These structures uncover the mechanism by which phosphorylation prevents caspase activity and also identify key regions of conformational control, which are functional allosteric sites. Second, various caspases can be inhibited by zinc, which has also been linked to apoptosis and Alzheimer's Disease. We are applying anomalous x-ray diffraction experiments to identify and characterize novel zinc-binding sites in caspases. Both of these approaches: phosphorylation and zinc-binding have helped us previously to identify new allosteric sites in caspases. By systematically applying these approaches, we can comprehensively map allosteric sites that are used across the caspase family as well as unique sites that are found only on one particular caspase. Our approaches are designed to provide the molecular details of allosteric control as well as assess the biological relevance of these mechanisms. The comparative map of caspase allostery by phosphorylation and zinc binding that we are generating will enable us to select the most appropriate regulatory sites for optimal control of caspase function and for effective treatment of diseases that involve caspases.